Biochemical markers of red blood cell storage and toxicity

ABSTRACT

Compositions and methods for determining post-transfusion survival or toxicity of red blood cells and the suitability of red blood cell units for transfusion by measuring the levels of one or more markers in a red blood cell sample are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/425,768, filed May 29, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/872,748, filed Jan. 16, 2018, now U.S. Pat. No.10,345,316, which is a continuation of U.S. patent application Ser. No.15/476,837, filed Mar. 31, 2017, now U.S. Pat. No. 9,903,876, which is acontinuation of U.S. patent application Ser. No. 14/012,885, filed Aug.28, 2013, now U.S. Pat. No. 9,638,687, which claims the benefit of U.S.Provisional Application No. 61/693,956, filed Aug. 28, 2012, each ofwhich is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. NIH R01HL092977, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD

The invention relates to compositions and methods for determiningpost-transfusion survival and toxicity of red blood cell (RBC) units bymeasuring the levels of one or more markers in a RBC sample.

BACKGROUND

In excess of 15,000,000 units of red blood cells (RBCs) are transfusedin the United States each year into an excess of 5,000,000 patients(approximately 1/70 Americans). Currently, there are only 3 qualitycontrol measures utilized prior to release of a unit of RBCs: 1) theabsence of screened pathogens, 2) compatibility with the patient, 3)storage history at 4° C. FDA guidelines for RBC storage require thatstored RBCs (up to 42 days) have less than 1% hemolysis and have 75% 24hour post-transfusion survival. However, it has been known for decadesthat RBCs store differently as a function of factors intrinsic to thedonor. For example, RBCs from some donors have only 30-40% 24 hourpost-transfusion survival, and this is consistently found with unitsfrom such donors. However, the factors that regulate whether the bloodfrom a given donor stores well or poorly is poorly understood. For thisreason, currently, there are no quality control measures regarding theextent to which a transfused unit of RBCs will survive post-transfusion.

This is a medical problem for two main reasons. First, RBCs that survivepoorly post-transfusion result in a less efficacious product from thestandpoint of RBC replacement. However, even more important is thenotion that RBCs that are cleared from circulation represent a toxicinsult to the recipient, which may result in morbidity and/or mortality.A second issue is what biochemical markers may predict RBCs that aregoing to be toxic from pathways other than simple RBC clearance. It hasbeen described that eicosanoids can accumulate in stored human RBCs, butthey can be difficult to detect.

There are currently no existing techniques to predict post-transfusionsurvival of RBC units or toxicity of said units. Thus, the presentdisclosure satisfies these and other needs. Disclosed herein is a methodfor assessing a RBC unit (prior to transfusion) allowing the predictionof its post-transfusion survival and toxicity. Specifically, biochemicalmarkers that predict if RBCs will survive well post-transfusion or willbe toxic are presented herein.

SUMMARY

Described herein are compositions and methods for determiningpost-transfusion survival and toxicity of a RBC unit by measuring thelevels of one or more markers in a RBC sample.

In a first aspect, disclosed herein is a method of determiningpost-transfusion survival of red blood cells (RBC) prior to transfusion,the method comprising the steps of: a) measuring the levels of one ormore markers in a RBC sample selected from the group consisting ofCytidine, 5-Methylcytidine, N4-acetylcytidine, 9-HODE, 13-HODE,Prostaglandin E2, 5-HETE, 12-HETE, 15-HETE, arachidonic acid,1-palmitoylglycerophosphoinositol, 1-stearoylglycerophosphoinositol, 13,14-dihydro-15-keto-prostaglandin E2, and the reticulocyte count of ablood donor at the time of donation; b) comparing the level of the oneor more markers in the RBC sample with the level of the one or moremarkers present in a control sample, wherein a higher or lower level ofthe one or more markers in the RBC sample is indicative of a lower RBCstorage quality.

In a second aspect, disclosed herein is a method of determining thesuitability of a red blood cell (RBC) unit for transfusion, the methodcomprising the steps of: a) measuring the levels of one or more markersin a RBC sample selected from the group consisting of Cytidine,5-Methylcytidine, N4-acetylcytidine, 9-HODE, 13-HODE, Prostaglandin E2,5-HETE, 12-HETE, 15-HETE, arachidonic acid,1-palmitoylglycerophosphoinositol, 1-stearoylglycerophosphoinositol, 13,14-dihydro-15-keto-prostaglandin E2, and the reticulocyte count of ablood donor at the time of donation; b) comparing the level of the oneor more markers in the RBC sample with the level of the one or moremarkers present in a control sample, wherein a higher or lower level ofthe one or more markers in the RBC sample is indicative of a lowersuitability for transfusion.

In various embodiments of the first and second aspects, the measurementis performed at the time of collection of the RBC sample.

In various embodiments of the first and second aspects, the measurementis performed during the time of storage of the RBC sample.

In various embodiments of the first and second aspects, the measurementis performed by mass spectrometry. In various embodiments, the massspectrometry is gas-chromatography/mass spectrometry (GC/MS) or liquidchromatography-tandem mass spectrometry (LC/MS/MS).

In various embodiments of the first and second aspects, the measurementis performed by enzymatic assay.

In various embodiments of the first and second aspects, the measurementis performed by ELISA.

In various embodiments of the first and second aspects, the level of theone or more marker is 2-200 fold higher than in the control sample.

In a third aspect, disclosed herein is method for determining RBCstorage quality, the method comprising the steps of: obtaining a datasetassociated with a sample of stored blood, wherein the dataset comprisesat least one marker, selected from the group consisting of Cytidine,5-Methylcytidine, N4-acetylcytidine, 9-HODE, 13-HODE, Prostaglandin E2,5-HETE, 12-HETE, 15-HETE, arachidonic acid,1-palmitoylglycerophosphoinositol, 1-stearoylglycerophosphoinositol, 13,14-dihydro-15-keto-prostaglandin E2, and the reticulocyte count of ablood donor at the time of donation; analyzing the dataset to determinedata for the at least one marker, wherein the data is positivelycorrelated or negatively correlated with RBC storage quality of thesample of stored blood.

In a fourth aspect, disclosed herein is method for determining RBCstorage quality, the method comprising the steps of: obtaining a sampleof stored blood, wherein the sample comprises at least one marker,selected from the group consisting of Cytidine, 5-Methylcytidine,N4-acetylcytidine, 9-HODE, 13-HODE, Prostaglandin E2, 5-HETE, 12-HETE,15-HETE, arachidonic acid, 1-palmitoylglycerophosphoinositol,1-stearoylglycerophosphoinositol, 13, 14-dihydro-15-keto-prostaglandinE2, and the reticulocyte count of a blood donor at the time of donation;contacting the sample with a reagent; generating a complex between thereagent and the at least one marker; detecting the complex to obtain adataset associated with the sample, wherein the dataset comprisesexpression or activity level data for the at least one marker; andanalyzing the expression or activity level data for the at least onemarker, wherein the expression or activity level of the at least onemarker is positively correlated or negatively correlated with RBCstorage quality.

In a fifth aspect, disclosed herein is computer-implemented method fordetermining RBC storage quality, the method comprising the steps of:storing, in a storage memory, a dataset associated with a stored bloodsample, wherein the dataset comprises data for at least one marker,selected from the group consisting of Cytidine, 5-Methylcytidine,N4-acetylcytidine, 9-HODE, 13-HODE, Prostaglandin E2, 5-HETE, 12-HETE,15-HETE, arachidonic acid, 1-palmitoylglycerophosphoinositol,1-stearoylglycerophosphoinositol, 13, 14-dihydro-15-keto-prostaglandinE2, and the reticulocyte count of a blood donor at the time of donation;and analyzing, by a computer processor, the dataset to determine theexpression or activity levels of the at least one marker, wherein theexpression or activity levels are positively correlated or negativelycorrelated with RBC storage quality.

In a sixth aspect, disclosed herein is system for determining RBCstorage quality, the system comprising: a storage memory for storing adataset associated with a stored blood sample, wherein the datasetcomprises data for at least one marker, wherein the dataset comprisesdata for at least one marker, selected from the group consisting ofCytidine, 5-Methylcytidine, N4-acetylcytidine, 9-HODE, 13-HODE,Prostaglandin E2, 5-HETE, 12-HETE, 15-HETE, arachidonic acid,1-palmitoylglycerophosphoinositol, 1-stearoylglycerophosphoinositol, 13,14-dihydro-15-keto-prostaglandin E2, and the reticulocyte count of ablood donor at the time of donation; and a processor communicativelycoupled to the storage memory for analyzing the dataset to determine theactivity or expression levels of the at least one marker, wherein theactivity or expression levels are positively correlated or negativelycorrelated with RBC storage quality.

In a seventh aspect, disclosed herein is computer-readable storagemedium storing computer-executable program code, the program codecomprising: program code for storing a dataset associated with a storedblood sample, wherein the dataset comprises data for at least onemarker, wherein the dataset comprises data for at least one marker,selected from the group consisting of Cytidine, 5-Methylcytidine,N4-acetylcytidine, 9-HODE, 13-HODE, Prostaglandin E2, 5-HETE, 12-HETE,15-HETE, arachidonic acid, 1-palmitoylglycerophosphoinositol,1-stearoylglycerophosphoinositol, 13, 14-dihydro-15-keto-prostaglandinE2, and the reticulocyte count of a blood donor at the time of donation;and program code for analyzing the dataset to determine the activity orexpression levels of the at least one marker, wherein the activity orexpression levels of the markers are positively correlated or negativelycorrelated with RBC storage quality.

In an eighth aspect, disclosed herein is method for predicting anegative transfusion outcome, the method comprising the steps of:obtaining a dataset associated with a sample of stored blood, whereinthe dataset comprises at least one marker, wherein the dataset comprisesdata for at least one marker, selected from the group consisting ofCytidine, 5-Methylcytidine, N4-acetylcytidine, 9-HODE, 13-HODE,Prostaglandin E2, 5-HETE, 12-HETE, 15-HETE, arachidonic acid,1-palmitoylglycerophosphoinositol, 1-stearoylglycerophosphoinositol, 13,14-dihydro-15-keto-prostaglandin E2, and the reticulocyte count of ablood donor at the time of donation; analyzing the dataset to determinedata for the at least one marker, wherein the data is positivelycorrelated or negatively correlated with a negative transfusion outcomeif the blood sample is transfused into a patient.

In a ninth aspect, disclosed herein is method for predicting a negativetransfusion outcome, the method comprising the steps of: obtaining asample of stored blood, wherein the sample comprises at least onemarker, wherein the dataset comprises data for at least one marker,selected from the group consisting of Cytidine, 5-Methylcytidine,N4-acetylcytidine, 9-HODE, 13-HODE, Prostaglandin E2, 5-HETE, 12-HETE,15-HETE, arachidonic acid, 1-palmitoylglycerophosphoinositol,1-stearoylglycerophosphoinositol, 13, 14-dihydro-15-keto-prostaglandinE2, and the reticulocyte count of a blood donor at the time of donation;contacting the sample with a reagent; generating a complex between thereagent and the at least one marker; detecting the complex to obtain adataset associated with the sample, wherein the dataset comprisesexpression or activity level data for the at least one marker; andanalyzing the expression or activity level data for the markers, whereinthe expression or activity level of the at least one marker ispositively correlated or negatively correlated with a negativetransfusion outcome if the blood sample is transfused into a patient.

In a tenth aspect, disclosed herein is computer-implemented method forpredicting a negative transfusion outcome, the method comprising thesteps of: storing, in a storage memory, a dataset associated with astored blood sample, wherein the dataset comprises data for at least onemarker wherein the dataset comprises data for at least one marker,selected from the group consisting of Cytidine, 5-Methylcytidine,N4-acetylcytidine, 9-HODE, 13-HODE, Prostaglandin E2, 5-HETE, 12-HETE,15-HETE, arachidonic acid, 1-palmitoylglycerophosphoinositol,1-stearoylglycerophosphoinositol, 13, 14-dihydro-15-keto-prostaglandinE2, and the reticulocyte count of a blood donor at the time of donation;and analyzing, by a computer processor, the dataset to determine theexpression or activity levels of the at least one marker, wherein theexpression or activity levels are positively correlated or negativelycorrelated with a negative transfusion outcome if the blood sample istransfused into a patient.

In an eleventh aspect, disclosed herein is system for predicting anegative transfusion outcome, the system comprising: a storage memoryfor storing a dataset associated with a stored blood sample, wherein thedataset comprises data for at least one marker, wherein the datasetcomprises data for at least one marker, selected from the groupconsisting of Cytidine, 5-Methylcytidine, N4-acetylcytidine, 9-HODE,13-HODE, Prostaglandin E2, 5-HETE, 12-HETE, 15-HETE, arachidonic acid,1-palmitoylglycerophosphoinositol, 1-stearoylglycerophosphoinositol, 13,14-dihydro-15-keto-prostaglandin E2, and the reticulocyte count of ablood donor at the time of donation; and a processor communicativelycoupled to the storage memory for analyzing the dataset to determine theactivity or expression levels of the at least one marker, wherein theactivity or expression levels are positively correlated or negativelycorrelated with a negative transfusion outcome if the blood sample istransfused into a patient.

In a twelfth aspect, disclosed herein is computer-readable storagemedium storing computer-executable program code, the program codecomprising: program code for storing a dataset associated with a storedblood sample, wherein the dataset comprises data for at least onemarker, selected from the group consisting of Cytidine,5-Methylcytidine, N4-acetylcytidine, 9-HODE, 13-HODE, Prostaglandin E2,5-HETE, 12-HETE, 15-HETE, arachidonic acid,1-palmitoylglycerophosphoinositol, 1-stearoylglycerophosphoinositol, 13,14-dihydro-15-keto-prostaglandin E2, and the reticulocyte count of ablood donor at the time of donation; and program code for analyzing thedataset to determine the activity or expression levels of the at leastone marker, wherein the activity or expression levels of the markers arepositively correlated or negatively correlated with a negativetransfusion outcome if the blood sample is transfused into a patient.

In various embodiments of the above aspects, the dataset is obtained atthe time of collection of the RBC sample.

In various embodiments of the above aspects, the dataset is obtainedduring the time of storage of the RBC sample.

In various embodiments of the above aspects, the dataset is obtained bymass spectrometry.

In various embodiments of the above aspects, the mass spectrometry isgas-chromatography/mass spectrometry (GC/MS) or liquidchromatography-tandem mass spectrometry (LC/MS/MS).

In various embodiments of the above aspects, the dataset is obtained byenzymatic assay.

In various embodiments of the above aspects, the dataset is obtained byELISA.

In a thirteenth aspect, disclosed herein is kit for use in predicting anegative transfusion outcome or red blood cell (RBC) storage quality,the kit comprising: a set of reagents comprising a plurality of reagentsfor determining from a stored blood sample data for at least one marker,selected from the group consisting of Cytidine, 5-Methylcytidine,N4-acetylcytidine, 9-HODE, 13-HODE, Prostaglandin E2, 5-HETE, 12-HETE,15-HETE, arachidonic acid, 1-palmitoylglycerophosphoinositol,1-stearoylglycerophosphoinositol, 13, 14-dihydro-15-keto-prostaglandinE2, and the reticulocyte count of a blood donor at the time of donation;and instructions for using the plurality of reagents to determine datafrom the stored blood sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E. Post-Transfusion Recovery of Stored Murine RBC units,Endogenous RBC Lifespan, and Metabolomic Analysis of Markers ofOxidative Stress During RBC storage. Murine RBCs were collected andstored (as described in Example 1) for 14 days, and then transfused intoH2-K^(b) GFP mice that express transgenic GFP on all hematopoieticcells, including RBCs. (FIG. 1A) Peripheral blood of recipient mice wassampled at indicated times post-transfusion and recovery of transfused,stored RBCs was determined by flow cytometric enumeration ofGFP-negative RBCs. (FIG. 1B) B6 or FVB mice underwent biotinylation invivo until 100% of their RBCs were avidin reactive. Peripheral bloodsamples were then stained with avidin-APC at the indicated time pointsand the percentage of avidin-reactive RBCs was enumerated, which alloweda determination of endogenous RBC lifespan. (FIGS. 1C-1E) Aliquots ofstored RBC units obtained from FVB and B6 mouse donors were frozen atthe indicated time points and were then analyzed by GC/MS and LC/MS/MS,as appropriate (as described in Example 1). Results are reported forproducts of lipid peroxidation (FIG. 1C), for glutathione pathwaycomponents (FIG. 1D), and for two natural anti-oxidants (FIG. 1E). Thesemetabolomic data represent the combined results from 3 separateexperiments; in each experiment, post-transfusion recovery wasdetermined at Day 14 of storage, with similar results to those in FIG.1A in each case.

FIG. 2. Metabolomic Analysis of Components of the Glutathione Synthesisand Degradation Pathway During Storage of Murine RBC Units. The samespecimens described in FIGS. 1C-1E, were analyzed for components in theglutathione synthesis pathway. These data represent the combined resultsfrom 3 separate experiments.

FIGS. 3A, 3B. Metabolomic Analysis of Components of the Glycolytic andPurine Metabolic Pathways During Storage of Murine RBC Units. The samespecimens described in FIGS. 1C-1E, were analyzed for components of theglycolytic (FIG. 3A) and purine metabolic (FIG. 3B). These datarepresent the combined results from 3 separate experiments.

FIG. 4. Arachidonic Acid and Eicosanoid Levels in Stored Murine RBCUnits, as Measured by Metabolomic Analysis. The same specimens describedin FIGS. 1C-1E were analyzed for determining levels of arachidonic acidand various eicosanoids. These data represent the combined results from3 separate experiments.

FIG. 5. Pyrimidine Metabolism in Murine RBC Units with DifferentialStorage Biology. The same murine specimens described in FIGS. 1A-1E wereanalyzed for cytidine metabolism (cytidine, 5-methylcytidine, andN4-acetylcytidine). These data represent the combined results from 3separate experiments.

DETAILED DESCRIPTION

The present invention generally relates to compositions and methods fordetermining post-transfusion survival and toxicity of RBCs by measuringthe levels of one or more markers in a RBC sample.

The invention described in this disclosure represents a method forassessing an RBC unit (prior to transfusion) allowing the prediction ofits post-transfusion survival and toxicity. Among the specific aims are:(1) Biochemical markers that predict if RBCs will survive wellpost-transfusion; and (2) Biochemical markers that predict if RBCs aretoxic post-transfusion.

Red blood cell (RBC) transfusion is a life-saving therapy, andrefrigerated storage is crucial for maintaining an adequate supply ofdonor units. However, recent studies have focused on potential adverseclinical sequelae resulting from transfusing humans with RBC unitsstored for longer periods of time. Indeed, multiple observationalstudies in human patients provide data demonstrating inferior clinicaloutcomes when older, stored RBC units are transfused¹. Nonetheless, thisissue remains controversial because other, similarly designed humanstudies, show no difference in clinical outcome when comparing patientsreceiving transfusions of older or fresher RBC units^(1,2). To begin toaddress this controversy, several prospective human trials are currentlyongoing, and one was recently completed³⁻⁵. However, it is notcontroversial that stored RBCs accumulate multiple factors that may betoxic when infused (e.g. microparticles, free iron, free hemoglobin,prostaglandins, and leukotrienes)⁶⁻⁴.

One complication in studying RBC transfusion is that there isconsiderable donor-to-donor variation in the effect of refrigeratedstorage on RBC function and quality. In addition, there is a generalabsence of robust analytic tests that consistently and accuratelypredict the quality of a given RBC unit prior to transfusion¹⁵. Due tothe genetic and environmental complexity of outbred human donorpopulations, and the difficulty in limiting the number of independentvariables in studying human RBC transfusion, we developed a robustanimal model to begin to address these issues¹⁶. Using inbred mousestrains in defined environmental and dietary settings limits theexperimental variability of the system, and allows for deliberatemanipulation of independent variables. This was combined withmetabolomic methods to determine whether variations in the levels,and/or changes in concentrations, of small molecules in vitro correlatedwith post-transfusion RBC recovery in vivo. In particular, we evaluatedwhether: 1) genetic background correlated with donor RBC storagequality, 2) metabolomic differences correlated with donor RBC storagequality, and 3) accumulation of potentially toxic molecules correlatedwith genetic background and/or donor RBC storage quality.

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only,and is not intended to be limiting. As used in this specification andthe appended claims, the singular forms “a”, “an” and “the” includeplural references unless the content clearly dictates otherwise.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein.

As used herein, “RBC storage quality” is defined as the extent ofpost-transfusion recovery of the stored RBCs; higher recovery is definedas higher quality. Examples of post-transfusion recovery include greaterthan zero and almost 100% recovery, i.e., recovery of 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, and all percentages in between. Inone embodiment, an acceptable RBC storage quality is an average of 75%post-transfusion recovery at 24 hours, as under FDA guidelines.

As used herein, “toxicity” of a RBC unit is defined as any adversereaction associated with transfusion of a RBC unit, including, but notlimited to, hemolytic transfusion reactions, exposure to freehemoglobin, iron overload, induction of recipient cytokines,introduction of procoagulant activity, and inhibition of recipientvascular relaxation, among others.

As used herein, a RBC unit is less suitable for transfusion if it haslower RBC quality (i.e., post-transfusion survival) or elevated toxicityas compared to other RBC units, e.g., as compared to a control.

An “analyte” or “target” refers to a compound to be detected. Suchcompounds can include small molecules, peptides, proteins, nucleicacids, as well as other chemical entities. In the context of the presentinvention, an analyte or target will generally correspond to thebiochemical compounds disclosed herein, or a reaction product thereof.

The term “biomarker” refers to a molecule (typically small molecule,protein, nucleic acid, carbohydrate, or lipid) that is expressed and/orreleased from a cell, which is useful for identification or prediction.Such biomarkers are molecules that can be differentially expressed,e.g., overexpressed or underexpressed, or differentially released inresponse to varying conditions (e.g., storage). In the context of thepresent invention, this frequently refers to the biochemical compoundsdisclosed herein, which are elevated in stored versus non-stored RBCs,for instance, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold or more in storedRBCs versus non-stored RBCs.

A “sample” refers to any source which is suspected of containing ananalyte or target molecule. Examples of samples which may be testedusing the present invention include, but are not limited to, blood,serum, plasma, urine, saliva, cerebrospinal fluid, lymph fluids, tissueand tissue and cell extracts, cell culture supernantants, among others.A sample can be suspended or dissolved in liquid materials such asbuffers, extractants, solvents, and the like. In the context of thepresent application, a sample is generally a stored RBC sample ofvarying length of storage.

“Antibody” refers to any immunoglobulin or intact molecule as well as tofragments thereof that bind to a specific epitope that may be used inthe practice of the present invention. Such antibodies include, but arenot limited to polyclonal, monoclonal, chimeric, humanized, singlechain, Fab, Fab′, F(ab)′ fragments and/or F(v) portions of the wholeantibody and variants thereof. All isotypes are encompassed by this termand may be used in the practice of this invention, including IgA, IgD,IgE, IgG, and IgM.

An “antibody fragment” refers specifically to an incomplete or isolatedportion of the full sequence of the antibody which retains the antigenbinding function of the parent antibody and may also be used in thepresent invention. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments.

An intact “antibody” for use in the invention comprises at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH₁, CH₂and CH₃. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or V_(L)) and a light chain constant region.The light chain constant region is comprised of one domain, C_(L). TheV_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxyl-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies can mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system. The term antibodyincludes antigen-binding portions of an intact antibody that retaincapacity to bind. Examples of binding include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., Nature, 341:544-546 (1989)),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR).

“Single chain antibodies” or “single chain Fv (scFv)” may also be usedin the present invention. This term refers to an antibody fusionmolecule of the two domains of the Fv fragment, V_(L) and V_(H).Although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see, e.g., Bird et al.,Science, 242:423-426 (1988); and Huston et al., Proc Natl Acad Sci USA,85:5879-5883 (1988)). Such single chain antibodies are included byreference to the term “antibody” fragments can be prepared byrecombinant techniques or enzymatic or chemical cleavage of intactantibodies.

A “monoclonal antibody” may be used in the present invention. Monoclonalantibodies are a preparation of antibody molecules of single molecularcomposition. A monoclonal antibody composition displays a single bindingspecificity and affinity for a particular epitope.

In one embodiment, the antibody or fragment is conjugated to an“effector” moiety. The effector moiety can be any number of molecules,including labeling moieties such as radioactive labels or fluorescentlabels.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thepeptide or used to detect antibodies specifically reactive with thepeptide.

Samples of RBCs stored for various amounts of time are compared to“control” samples which can be freshly drawn RBCs or RBCs which havebeen minimally stored. Control samples are assigned a relative analyteamount or activity to which sample values are compared. Relevant levelsof analyte elevation occur when the sample amount or activity valuerelative to the control is 110%, more preferably 150%, more preferably200-500% (i.e., two to five fold higher relative to the control), morepreferably 1000-3000% higher.

Assays for many of the biochemical compounds disclosed herein are knownor commercially available.

For example, antibody reagents can be used in assays to detect thelevels of analytes in RBC samples using any of a number of immunoassaysknown to those skilled in the art.

Immunoassay techniques and protocols are generally described in Priceand Newman, “Principles and Practice of Immunoassay,” 2nd Edition,Grove's Dictionaries, 1997; and Gosling, “Immunoassays: A PracticalApproach,” Oxford University Press, 2000. A variety of immunoassaytechniques, including competitive and non-competitive immunoassays, canbe used. See, e.g., Self et al., Curr. Opin. Biotechnol., 7:60-65(1996). The term immunoassay encompasses techniques including, withoutlimitation, enzyme immunoassays (EIA) such as enzyme multipliedimmunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA),IgM antibody capture ELISA (MAC ELISA), and microparticle enzymeimmunoassay (META); immunohistochemical (IHC) assays; capillaryelectrophoresis immunoassays (CEIA); radioimmunoassays (RIA);immunoradiometric assays (IRMA); fluorescence polarization immunoassays(FPIA); and chemiluminescence assays (CL). If desired, such immunoassayscan be automated. Immunoassays can also be used in conjunction withlaser induced fluorescence. See, e.g., Schmalzing et al.,Electrophoresis, 18:2184-93 (1997); Bao, J. Chromatogr. B. Biomed. Sci.,699:463-80 (1997). Liposome immunoassays, such as flow-injectionliposome immunoassays and liposome immunosensors, are also suitable foruse in the present invention. See, e.g., Rongen et al., J. Immunol.Methods, 204:105-133 (1997). In addition, nephelometry assays, in whichthe formation of protein/antibody complexes results in increased lightscatter that is converted to a peak rate signal as a function of themarker concentration, are suitable for use in the methods of the presentinvention. Nephelometry assays are commercially available from BeckmanCoulter (Brea, Calif.; Kit #449430) and can be performed using a BehringNephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biochem.,27:261-276 (1989)).

Specific immunological binding of the antibody to proteins can bedetected directly or indirectly. Direct labels include fluorescent orluminescent tags, metals, dyes, radionuclides, and the like, attached tothe antibody. A chemiluminescence assay using a chemiluminescentantibody specific for the protein is suitable for sensitive,non-radioactive detection of protein levels. An antibody labeled withfluorochrome is also suitable. Examples of fluorochromes include,without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin,B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and lissamine.Indirect labels include various enzymes well known in the art, such ashorseradish peroxidase (HRP), alkaline phosphatase (AP),β-galactosidase, urease, and the like. A horseradish-peroxidasedetection system can be used, for example, with the chromogenicsubstrate tetramethylbenzidine (TMB), which yields a soluble product inthe presence of hydrogen peroxide that is detectable at 450 nm. Analkaline phosphatase detection system can be used with the chromogenicsubstrate p-nitrophenyl phosphate, for example, which yields a solubleproduct readily detectable at 405 nm. Similarly, a β-galactosidasedetection system can be used with the chromogenic substrateo-nitrophenyl-β-D-galactopyranoside (ONPG), which yields a solubleproduct detectable at 410 nm. An urease detection system can be usedwith a substrate such as urea-bromocresol purple (Sigma Immunochemicals;St. Louis, Mo.).

A signal from the direct or indirect label can be analyzed, for example,using a spectrophotometer to detect color from a chromogenic substrate;a radiation counter to detect radiation such as a gamma counter fordetection of ¹²⁵I; or a fluorometer to detect fluorescence in thepresence of light of a certain wavelength. For detection ofenzyme-linked antibodies, a quantitative analysis can be made using aspectrophotometer such as an EMax® Microplate Reader (Molecular Devices;Menlo Park, Calif.) in accordance with the manufacturer's instructions.If desired, the assays of the present invention can be automated orperformed robotically, and the signal from multiple samples can bedetected simultaneously.

The antibodies can be immobilized onto a variety of solid supports, suchas magnetic or chromatographic matrix particles, the surface of an assayplate (e.g., microtiter wells), pieces of a solid substrate material ormembrane (e.g., plastic, nylon, paper), and the like. An assay strip canbe prepared by coating the antibody or a plurality of antibodies in anarray on a solid support. This strip can then be dipped into the testsample and processed quickly through washes and detection steps togenerate a measurable signal, such as a colored spot.

In some embodiments, the measurement of the markers of the presentinvention is performed using various mass spectrometry methods. As usedherein, the term “mass spectrometry” or “MS” refers to an analyticaltechnique to identify compounds by their mass. MS refers to methods offiltering, detecting, and measuring ions based on their mass-to-chargeratio, or “m/z”. MS technology generally includes (1) ionizing thecompounds to form charged compounds; and (2) detecting the molecularweight of the charged compounds and calculating a mass-to-charge ratio.The compounds may be ionized and detected by any suitable means. A “massspectrometer” generally includes an ionizer and an ion detector. Ingeneral, one or more molecules of interest are ionized, and the ions aresubsequently introduced into a mass spectrographic instrument where, dueto a combination of magnetic and electric fields, the ions follow a pathin space that is dependent upon mass (“m”) and charge (“z”). See, e.g.,U.S. Pat. No. 6,204,500, entitled “Mass Spectrometry From Surfaces;”U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem MassSpectrometry;” U.S. Pat. No. 6,268,144, entitled “DNA Diagnostics BasedOn Mass Spectrometry;” U.S. Pat. No. 6,124,137, entitled“Surface-Enhanced Photolabile Attachment And Release For Desorption AndDetection Of Analytes;” Wright et al., Prostate Cancer and ProstaticDiseases 1999, 2: 264-76; and Merchant and Weinberger, Electrophoresis2000, 21; 1164-67.

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and turbulent flow liquidchromatography (TFLC) (sometimes known as high turbulence liquidchromatography (HTLC) or high throughput liquid chromatography).

In some embodiments, the present invention is practiced using computerimplementation. In one embodiment, a computer comprises at least oneprocessor coupled to a chipset. Also coupled to the chipset are amemory, a storage device, a keyboard, a graphics adapter, a pointingdevice, and a network adapter. A display is coupled to the graphicsadapter. In one embodiment, the functionality of the chipset is providedby a memory controller hub and an I/O controller hub. In anotherembodiment, the memory is coupled directly to the processor instead ofthe chipset.

The storage device is any device capable of holding data, like a harddrive, compact disk read-only memory (CD-ROM), DVD, or a solid-statememory device. The memory holds instructions and data used by theprocessor. The pointing device may be a mouse, track ball, or other typeof pointing device, and is used in combination with the keyboard toinput data into the computer system. The graphics adapter displaysimages and other information on the display. The network adapter couplesthe computer system to a local or wide area network.

As is known in the art, a computer can have different and/or othercomponents than those described previously. In addition, the computercan lack certain components. Moreover, the storage device can be localand/or remote from the computer (such as embodied within a storage areanetwork (SAN)).

As is known in the art, the computer is adapted to execute computerprogram modules for providing functionality described herein. As usedherein, the term “module” refers to computer program logic utilized toprovide the specified functionality. Thus, a module can be implementedin hardware, firmware, and/or software. In one embodiment, programmodules are stored on the storage device, loaded into the memory, andexecuted by the processor.

Embodiments of the entities described herein can include other and/ordifferent modules than the ones described here. In addition, thefunctionality attributed to the modules can be performed by other ordifferent modules in other embodiments. Moreover, this descriptionoccasionally omits the term “module” for purposes of clarity andconvenience.

The following examples of specific aspects for carrying out the presentinvention are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way.

EXAMPLES Example 1: Materials and Methods Collection and Processing ofMouse RBC Units

H2-K^(b) GFP+ mice were a generous gift from Dr. Derek A. Persons (St.Jude Children's Research Hospital; Memphis, Tenn.) and were bred by theEmory University Division of Animal Husbandry by crossing heterozygousmice with C57BL/6J mice in excess of 10 generations¹⁷; the RBCs of thesemice express Green Fluorescent Protein (GFP) and are readily enumeratedby flow cytometry. These mice were used as the universal transfusionrecipients in all of the described studies. C57BL/6J and FVB/J mice werepurchased from The Jackson Laboratory (Bar Harbor, Me.). All mice werehoused in identical conditions with identical diets and identical accessto water. In an effort to model human RBC storage, mouse donor RBCs werecollected and stored in a similar fashion to human RBC storage, aspreviously described¹⁶. All studies were carried out under protocolsapproved by the Institutional Animal Care and Use Committees.

Assessment of Post-Transfusion RBC Recovery and Endogenous RBC Lifespan

For all RBC storage experiments, donor mouse RBCs were stored at 4° C.for 14 days; this time frame was previously identified, using C57/BL6Jmouse donor RBCs, as appropriately approximating the refrigerated shelflife identified by the Food and Drug Administration for human RBCs; thatis, on average, 75% of donor mouse RBCs were still circulating 24 hrpost-transfusion at the Day 14 “outdate. ¹⁶” Therefore, at Day 14 ofstorage, 100 μl of stored, donor, packed RBCs (i.e. one mouse “unit”)were transfused into H2-K^(b) GFP+ recipient mice. At 10 min, 30 min, 1hr, 4 hr, and 24 hr post-transfusion, peripheral blood was obtained fromrecipients, and transfused RBCs were enumerated by flow cytometry bygating on GFP-negative RBC events. Peripheral blood obtained fromnon-transfused mice was used to enumerate the low number of events inthe GFP-negative gate, which were then subtracted from the analysis oftransfused RBCs.

For determination of endogenous RBC lifespan, mice received 3 dailyinjections of NHS-biotin i.p. (Pierce, Thermo Scientific) until ˜100% ofcirculating RBCs were reactive with avidin-allophycocyanin, as assessedby flow cytometry. Peripheral RBCs were then obtained weekly and stainedwith avidin-allophycocyanin, followed by enumeration of positive andnegative RBCs by flow cytometry. These data were then plotted todetermine RBC lifespan.

Mass Spectrometry Analysis of RBC Samples

Donor RBC samples, freshly obtained and at various times afterrefrigerated storage, were rapidly frozen using dry ice/ethanol andstored at 80° C. The supernatant was not stored separately nor were theRBCs washed and stored separately; thus, the results obtained evaluatedthe metabolites in the entire “unit.” Samples were shipped on dry ice toMetabolon Inc., where they were split into equal parts for analysis bygas-chromatography/mass spectrometry (GC/MS) and liquidchromatography-tandem mass spectrometry (LC/MS/MS). The LC/MS/MSplatform was based on a Waters ACQUITY UPLC and a Thermo-Finnigan LTQmass spectrometer, which consisted of an electrospray ionization (ESI)source and linear ion-trap (LIT) mass analyzer. The sample extract wassplit into two aliquots, dried, and then reconstituted in acidic orbasic LC-compatible solvents, each of which contained 11 or moreinjection standards at fixed concentrations. One aliquot was analyzedusing acidic positive-ion optimized conditions and the other using basicnegative-ion optimized conditions in two independent injections usingseparate dedicated columns. Extracts reconstituted in acidic conditionswere gradient eluted using water and methanol, both containing 0.1%Formic acid, whereas the basic extracts, which also used water/methanol,contained 6.5 mM Ammonium Bicarbonate. The MS analysis alternatedbetween MS and data-dependent MS² scans using dynamic exclusion. Thesamples destined for GC/MS analysis were re-dried under vacuumdesiccation for a minimum of 24 hr prior to being derivatized underdried nitrogen using bistrimethyl-silyl-triflouroacetamide. The GCcolumn was 5% phenyl and the temperature ramp was from 40° to 300° C. ina 16 minute period. Samples were analyzed on a Thermo-Finnigan Trace DSQfast-scanning single-quadrupole mass spectrometer using electron impactionization. Compounds were identified by comparison to library entriesof purified standards or recurrent unknown entities. Identification ofknown chemical entities was based on comparison to metabolomic libraryentries of purified standards. As of the time of analysis, more than1000 commercially-available purified standard compounds had beenacquired and registered into LIMS for distribution to both the LC and GCplatforms for determination of their analytical characteristics. Thecombination of chromatographic properties and mass spectra gave anindication of a match to the specific compound or an isobaric entity.

The peak areas for each identified biochemical entity were logtransformed, scaled to the median value for each compound observed inthe experiment, and normalized to Bradford protein content; resultsbelow the limit of detection were imputed with the minimum observedvalue for the compound. A Two-Way ANOVA with Contrasts was used todetermine the significance of variable main effects (e.g. Condition orTime/Day) and their interaction, and to identify biochemical entitiesthat differed significantly between experimental groups (p<0.05). Anestimate of the false discovery rate (q-value) is calculated to takeinto account the multiple comparisons that normally occur inmetabolomic-based studies.

Example 2: Variation Among Inbred Mouse Strains in Post-Transfusion RBCRecovery after Refrigerated Storage

As used herein, “RBC storage quality” is defined as the extent ofpost-transfusion recovery of the stored RBCs; higher recovery is definedas higher quality. Thus, to test the hypothesis that donor-specificvariation in RBC quality after refrigerated storage exists in non-humansystems, mice were chosen as the experimental model. After screening 6common inbred laboratory strains of mice from Jackson labs (C57BL6/J,FVB/J, C3H/J, BALBc/J, SJL/J, DBA2/J) and observing variation in RBCstorage quality between strains (data not shown), we focused our studieson a direct comparison of the one that exhibited the best storagequality (C57BL/6J; i.e. B6), and the one that exhibited the worst RBCstorage quality (FVB/NJ; i.e. FVB). To this end, peripheral blood RBCswere obtained from B6 and FVB mice, filter leukoreduced, stored for 14days, transfused, and evaluated for post-transfusion recovery using amethod that models human RBC storage¹⁶; these prior studies demonstratedthat 14 days of storage provided results with B6 RBCs that mimickedthose found with healthy human blood donors after 42 days of storage(i.e. ˜75% post-transfusion recovery). To allow post-transfusiontracking without labeling or otherwise modifying the donor RBCs, asingle strain of recipient mice was utilized that expressed transgenicGFP in their RBCs; in this way, no artifactual damage to the donor RBCswas introduced by any further pre-transfusion manipulation or anylabeling process. The circulating, transfused donor RBCs were thenquantified by flow cytometry by gating on the GFP-negative population.

RBCs from each donor strain were stored at the same hematocrit, and thesame volume of RBCs was transfused from each strain into naïverecipients. All donor RBCs were transfused intravenously through thetail vein. As is typically the case when transfusing stored human ormurine RBCs into either human or murine recipients, respectively, therewas a rapid clearance phase followed by a significantly slower clearancephase (FIG. 1A). The early phase clearance in humans has beeninterpreted as the rapid extravascular clearance of significantlystorage-damaged RBC¹⁸, which has been demonstrated to be the case inmice, since depletion of phagocytes prevents the early clearance fromoccurring¹¹. At 10 min post-transfusion, approximately 40% fewertransfused FVB RBCs were still circulating in peripheral blood, ascompared with transfused B6 RBCs (FIG. 1A). This statisticallysignificant difference persisted over time, with FVB RBCs having only˜50% of the 24-hour post-transfusion recovery of B6 RBCs (FIG. 1A). Thepost-transfusion recovery of 14-day old B6 RBCs was comparable to thatseen previously¹⁶. Taken together, these data demonstrate astrain-specific difference in donor RBC storage quality and suggest thata genetic difference(s) may account for the observed effects.

To test if RBC quality after refrigerator storage in vitro correlatedwith the endogenous RBC lifespan in the donor mice in vivo, the totalblood volumes of B6 and FVB mice were each labeled by biotinylation invivo. In this method, any new RBCs that are generated do not have biotinon their surface; thus, RBC lifespan can be determined by the rate ofdecrease of avidin-reactive RBCs over time¹⁹. Using this approach, FVBRBCs had a shorter endogenous lifespan as compared to B6 RBCs,correlating with the trend seen in refrigerated RBC storage quality(FIG. 1B). However, the natural differences in endogenous lifespan werenot as pronounced as the differences in post-transfusion recovery ofstored RBCs. Taken together, these data demonstrate strain-dependentdifferences in endogenous RBC lifespan, which correlate with amplifieddifferences in post-transfusion recovery resulting from refrigerated RBCstorage.

Example 3: Analysis of Anti-Oxidant Pathways and Accumulation ofOxidative Damage During RBC Storage

Mass spectrometric analysis captured multiple types of metabolites thatwere tracked during storage. Disclosed herein are several illustrativetypes of metabolites and their associated biosynthetic and catabolicpathways. Levels of 9,10 epoxystearate (as an indicator of lipidoxidation) increased progressively in refrigerator-stored FVB RBC units,but remained largely at baseline levels in stored B6 RBC units (FIG.1C). Likewise, oxidation of cholesterol into both7-alpha-hydroxycholesterol and 7-beta-hydroxycholesterol, and theirsubsequent conversion into 7-ketocholesterol, progressively increased inFVB RBC units, but not in B6 units (FIG. 1C). However, cholesterollevels in both FVB and B6 units did not differ and did not changesignificantly with storage time. Taken together, these data demonstratethat increased lipid oxidation correlated with poor storage quality ofFVB RBCs.

Reduced glutathione (GSH) is generally considered to be the most robustanti-oxidant in RBCs. Although no statistically significant differencesin GSH levels were observed when comparing stored RBC units from B6 andFVB mice (FIG. 1D), B6 RBC units had consistently higher levels ofoxidized glutathione (GSSG) and cysteine-glutathione disulfides (FIG.1D); the latter also increased progressively with storage in B6 units.In addition, although levels of the natural anti-oxidantalpha-tocopherol (i.e. Vitamin E) were equivalent at the time of bloodcollection, alpha-tocopherol levels dropped rapidly in FVB, but not B6,RBC units (FIG. 1E). Moreover, levels of another anti-oxidant,ergothioneine, were significantly higher on the day of collection in B6RBC units, as compared to FVB RBC units (FIG. 1E); these levels alsoincreased during storage in B6, but not FVB, RBC units.

Analysis of multiple components of the GSH biosynthetic pathway revealedthat B6 RBC units accumulated higher levels of homocysteine and cysteineduring storage, as compared to FVB RBC units (FIG. 2); these couldpotentially provide more substrate for GSH synthesis de novo in the RBCunits by combining with glutamate and glycine. Nonetheless, nodifferences in glutamate or glycine levels were observed when comparingFVB and B6 RBC units, either at baseline or during storage. In addition,the sum of GSH and GSSG levels was higher in B6, as compared to FVB, RBCunits. Finally, as compared with FVB RBC units, B6 RBC units hadsignificantly higher levels of byproducts of GSH synthesis, including2-hydroxybutyrate, 2-aminobutyrate, and ophthalmate (FIG. 2).Interestingly, accumulation of ophthalmate was previously reported toreflect GSH consumption in an effort to ameliorate oxidative stress²⁰.Taken together, these results suggest that there may be greater fluxthrough the GSH pathway in B6 RBC units, and that RBC storage qualitycorrelates positively with an enhanced ability to handle oxidativestress.

Example 4: Evaluation of Glycolysis and Purine Metabolism During RBCStorage

When glycolytic pathways were analyzed (FIG. 3A), the results weresimilar to what has been repeatedly reported regarding refrigeratedstorage of human RBCs⁶. In particular, glucose and 2,3-DPG levelsprogressively declined, whereas lactate levels progressively increasedin stored RBC units from both mouse strains. In contrast, pyruvatelevels increase and remain elevated during storage of B6, as compared toFVB, RBC units.

Because adenine is a component of various RBC storage solutions (e.g.CPDA-1), its initial levels in RBC units are high due to thissupplementation. During refrigerated storage, adenine progressivelydeclined, and AMP levels slowly increased, similarly in both B6 and FVBRBC units (FIG. 3B). Inosine, into which adenine can be converted,increased steadily over storage time in RBC units from both mousestrains, but with higher levels in FVB RBC units. Both hypoxanthine andxanthine increased significantly over time, with higher endpoint levelsin FVB, as compared to B6, RBC units; these results suggest that asubstantial quantity of inosine can be metabolized in a way thatprevents subsequent ATP synthesis. Urate levels were also higher in B6,as compared to FVB, RBC units, and levels of allantoin (the mainbreakdown product of urate) remained relatively stable in both B6 andFVB RBC units.

Example 5: Accumulation of Eicosanoids During RBC Storage

The RBC membrane is a rich source of phospholipids, and the oxidationand/or enzymatic processing of these can produce arachidonic acid,which, in turn, can be converted into prostaglandins and/or leukotrienes(the latter, together, are termed eicosanoids). Eicosanoids have potentand widespread effects on inflammation, vascular tone, vascularpermeability, and platelet activation²¹⁻²³. Interestingly, arachidonicacid (or, equivalently, arachidonate) accumulated progressively duringstorage of both B6 and FVB RBC units (FIG. 4). Although baseline levelswere significantly higher in FVB RBC units, peak levels at Day 14 wereequivalent in both strains. The source of arachidonic acid in thissetting is likely to be phospholipase-induced release of fatty acidsfrom the sn-2 position of inositol-containing phospholipids. However,unlike the similar levels of arachidonic acid, there was a substantialincrease in lysophospholipid by-products (i.e.1-palmitoylglycerophosphoinositol and 1-stearoylglycerophosphoinositol)in FVB, but not B6, RBC units. There was also a dramatic accumulation ofeicosanoids during storage of FVB, but not B6, RBC units. The latterincluded prostaglandin E2 and several leukotriene precursors (i.e.5-HETE, 12-HETE, and 15-HETE). In addition, in FVB RBC units, the levelsof linoleic acid metabolites (i.e. 13-HODE and 9-HODE) significantlyincreased during storage; in contrast, these remained low in B6 RBCunits. Together, these data suggest that there is increased generationand metabolism of arachidonic acid in FVB RBC units, with concomitantincreases in the synthesis of multiple eicosanoids during storage ofthese units.

Example 6: Pyrimidine Metabolism During RBC Storage

Assessment of pyrimidine metabolism demonstrated that FVB RBC units hadhigher levels of cytidine at baseline, as compared to those obtainedfrom B6 mice (FIG. 5). In addition, during storage, cytidine levelsincreased to a significantly greater extent in FVB, as compared to B6,RBC units. Levels of N4-acetylcytidine (a cytidine metabolite) were alsoapproximately 8-fold higher, at all time points measured, in FVBcompared to B6 RBC units (FIG. 5). Finally, the levels of one downstreammetabolite, uracil, were significantly greater in FVB than B6 RBC unitsat end of storage; however, no differences were observed in the levelsanother metabolite, 5-methylcytidine.

Example 7: Application of Markers as a Diagnostic Test of RBCs

As a result of the studies above, we have identified a distinct panel ofmetabolites that predicts how well stored RBCs will survivepost-transfusion. This panel includes members of pyrimidine metabolism,cytidine and its breakdown products. These include: Cytidine,5-Methylcytidine, N4-acetylcytidine. A further panel includes markersthat indicate the generation of inflammatory eicosanoids. These include:9-HODE, 13-HODE, Prostaglandin E2, 5-HETE, 12-HETE, 15-HETE, arachidonicacid, 1-palmitoylglycerophosphoinositol,1-stearoylglycerophosphoinositol, and 13,14-dihydro-15-keto-prostaglandin E2.

The above markers of RBC unit quality may be applied to evaluation ofRBC units in several different ways. First, a sample of an RBC unit canbe subjected to mass spectrometry and the profile of the above markerscan be generated (all from a single sample). This profile would then beused to predict the post-transfusion survival of the RBC unit. Suchinformation would allow two distinct medical advantages: 1) direction ofbetter units of RBCs to patients whose disease status makes themparticularly susceptible to toxicity of poorly stored RBC units, and 2)management of the blood supply such that storage time for individualunits could be tailored to the biology of the unit. Moreover, it ispossible that FDA guidelines could be tailored to utilize these specificchemical measurements as release criteria for RBC units. In oneembodiment, a high throughput mass spectrometer is used. Alternatively,individual assays could be run on a much smaller platform by traditionalassay techniques (i.e. ELISA, enzymatic assay, etc.). Such would allow asimplified platform with a less expensive instrumentation. For suchpurposes, a small number of the above chemical entities that wererepresentative of the whole would be identified and measured.

With respect to cytidine as a marker, it is noted that the in vivolifespan of FVB RBCs was shorter than that of C57BL/6 (B6), whichcorrelates with the trend in RBC storage biology—FVB RBCs store morepoorly than do B6 RBCs. In addition, it is observed that the FVB RBCsthat store more poorly have increased levels of cytidine, and thiscorrelation was also observed on human RBCs. Based upon the new in vivodata, we raise the possibility that FVB mice had higher reticulocytecounts than did B6, as a compensatory mechanism for shorter RBClifespan, so as to maintain normal hematocrit levels. Sincereticulocytes are a rich source of RNA, which can then be broken downinto cytidine, this constitutes a novel explanatory hypothesis for theobserved biology.

Patients whose RBCs store poorly have shorter natural in vivo RBClifespan (as a result of their intrinsic biology). As a result ofshorter RBC lifespan, they make RBCs more quickly, and thus have higherreticulocyte counts. As a result of higher reticulocyte count, they havehigher RNA in the unit. As a result of higher RNA in the unit, they havehigher levels of cytidine. This explanation suggests that cytidine is acorrelative marker of someone who has RBCs with an intrinsically shorterlifespan. This does not speak to mechanism, and suggests that cytidineis just a correlative marker. However, if this association turns out tobe true, then the reticulocyte count itself should be equallypredictive. Thus, this line of reasoning leads to the following aim.Measuring reticulocyte count of a blood donor at the time of donationwill predict how well the RBCs will store and how well they willcirculate after transfusion.

Thus, reticulocyte counts, which are routinely determined in thehospital, can be used as an indicator of RBC storage quality. Inparticular, patients with underlying pathology for which it would bepredicted that poorly storing RBCs would be particularly dangerous wouldselectively get RBCs from donors that had low reticulocyte count at timeof donation.

DISCUSSION

The data presented herein demonstrate that different inbred strains ofmice (i.e. B6 and FVB) have significant differences in RBC storagequality (defined as post-transfusion RBC recovery), which is the goldstandard for evaluating the storage of human RBC donor units. Becausepost-transfusion RBC clearance depends on the storage quality of theRBCs themselves, as well as on the activity of the recipient'smononuclear phagocyte system, we used one mouse strain as the universaltransfusion recipient for these studies (i.e. GFP transgenic mice on aB6 background). This experimental design decreases concerns regardingdifferences in RBC clearance biology in the recipient, allowing anexclusive focus on the storage of the donor RBC units. Although thisapproach could raise the concern that the B6 transfusions weresyngeneic, whereas the FVB transfusions were allogeneic, the 24-hr RBCrecovery assay is complete long before adaptive immunity is induced. Inaddition, B6 mice have no “naturally-occurring” antibodies thatrecognize FVB RBC antigens as tested by flow cytometry, both in vitroand in vivo (data not shown). Moreover, there is no difference whenB6xFVB F1 recipients are used, the storage quality of B6 RBCs issignificantly better than FVB RBCs (data not shown). Finally, thedescribed accumulation of oxidative damage (FIGS. 1A-1E) and eicosanoids(FIG. 4) occurs in vitro during storage prior to transfusion; thus,these effects are unrelated to potential allogenicity in vivo. Takentogether, these data describe strain-specific variations in donor RBCstorage quality, and suggest that this variation is caused, at least inpart, by genetic determinants.

The current results with stored mouse RBC units suggest that oxidativedamage increases with storage duration, particularly for FVB mice (FIG.1C). In addition, levels of two endogenous anti-oxidants (i.e.alpha-tocopherol and ergothioneine) are maintained at higher levels inB6, as compared to FVB, stored RBCs units (FIG. 1E). Although theresults for the GSH biosynthetic pathway are more complex (FIG. 1D andFIG. 2), one explanation that is consistent with the current data positsthat synthesis and flux through this pathway is greater in stored B6 RBCunits, thereby allowing these units to handle oxidative stress moreeffectively than those obtained from FVB mice. In addition, the findingspresented in FIGS. 1A-1E and 2 are consistent with a previous report ofoxidative stress-induced hemolysis in inbred mouse strains; in thissetting, B6 RBCs exhibited decreased H₂02-induced injury, as compared toRBCs obtained from other strains; however, FVB mice were not studied²⁴.Finally, an important role for oxidative stress and glutathione in thebiology of RBC storage has been described previously, particularly withregard to human RBC storage²⁵⁻²⁷.

Recently, eicosanoids have been reported to accumulate during storage ofhuman RBC units. The same molecular species as we observed in FVB RBCunits were reported in human units (i.e. Arachidonic acid, 5-HETE,12-HETE, and 15-HETE)¹³; however, prostaglandin increase was not noted.To the best of our knowledge, the work disclosed herein is the firstdemonstration of substantial donor variation (in mice) regarding theaccumulation of eicosanoids during RBC storage, and also the firstindication that this variation may have a genetic basis in any species.In FVB RBC units, the maintenance of arachidonic acid levels duringstorage, in the context of increasing levels of lysophospholipids andeicosanoids, suggests a rapid flux through this pathway (in terms ofarachidonic acid generation and its subsequent conversion intodownstream products). In contrast, although arachidonic acid levelsincrease during storage of B6 RBC units, there is little generation oflysophospholipids or conversion to eicosanoids. Taken together, theseresults suggest the testable hypothesis that FVB RBC units have higherphospholipase, cyclooxygenase, and lipoxygenase activities, as comparedto B6 RBC units. It is worth noting that C57BL/6 mice are deficient in asecreted phospholipase A2 (sPLA2), which may explain increasedarachidonic acid generation in FVB mice. However, this would not explainthe increased conversion into prostaglandins and leukotrienes, whichwould require other differences in cyclooxygenase and lipoxygenaseactivities. Of particular note is that analysis of human RBC units havenot detected cellular or soluble phospholipase activity (cPLA2 and sPLA2were not detected)²⁸. Rather, an alternate source of phospholipase hasbeen implicated, in particular, peroxiredoxin-6²⁸.

While the above changes have been noted in “stored RBC units”, neitherthe location (e.g. cellular or supernatant) nor the source (e.g. theRBCs themselves, residual leukocytes, or contaminating platelets) isdetermined by the current methodologies. Although leukoreduction resultsin a unit with essentially undetectable leukocytes, it seems likely thatsome remain. Moreover, platelets are far more numerous in mice than inhumans, and while leukoreduction does decrease murine platelets in RBCunits, significant numbers of detectable platelets persist (data notshown). Accordingly, while RBCs may turn out to be the source ofeicosanoids, it would be premature and potentially incorrect to draw anyinferences that the RBCs are the source of eicosanoids from the currentstudies. Nevertheless, regardless of the cellular source, theobservation of dramatic differences between genetically distinct strainspersists.

Long-term dietary and/or environmental factors could affect RBC storagequality. Indeed, the results of the classical human studies of Dern etal. could be due to heritable factors or long-term dietary behaviorand/or environmental exposure²⁹. In the current study, control overvariation in acquired factors which may influence RBC storage wasattempted in a controlled laboratory environment. All animals werefemales of the same age, were provided the same commercial mouse chow adlibitum and were housed in the same vivarium with identical sources ofbedding, water, and caging.

SUMMARY

In summary, RBC transfusion is a life-saving therapy, the logisticalimplementation of which requires RBC storage. However, stored RBCsexhibit substantial donor variability in multiple characteristics,including hemolysis in vitro and RBC recovery in vivo. The basis ofdonor variability is poorly understood.

We applied a murine model of RBC storage and transfusion to test thehypothesis that genetically distinct inbred strains of mice woulddemonstrate strain-specific differences in RBC storage. In vivorecoveries were determined by monitoring transfused RBCs over 24 hours.Timed aliquots of stored RBCs were subjected to tandemchromatography/mass spectrometry analysis to elucidate metabolic changesin the RBCs during storage.

Using independent inbred mouse strains as donors, we found substantialstrain-specific differences in post-transfusion RBC recovery in vivofollowing standardized refrigerated storage in vitro. Poorpost-transfusion RBC recovery correlated with reproducible metabolicvariations in the stored RBC units, including increased lipidperoxidation, decreased levels of multiple natural antioxidants, andaccumulation of cytidine. Strain-dependent differences were alsoobserved in eicosanoid generation (i.e. prostaglandins andleukotrienes).

These findings provide the first evidence of strain-specific metabolomicdifferences following refrigerated storage of murine RBCs. They alsoprovide the first definitive biochemical evidence for strain specificvariation of eicosanoid generation during RBC storage. The moleculesdescribed that correlate with RBC storage quality, and their associatedbiochemical pathways, suggest multiple causal hypotheses that can betested regarding predicting the quality of RBC units prior totransfusion and developing methods of improved RBC storage.

REFERENCES

-   1. van de Watering L. Red cell storage and prognosis. Vox Sang 2011;    100: 36-45.-   2. van de Watering L. Pitfalls in the current published    observational literature on the effects of red blood cell storage.    Transfusion 2011; 51: 1847-1854.-   3. Fergusson D A, Hebert P, Hogan D L, LeBel L, Rouvinez-Bouali N,    Smyth J A, Sankaran K, Tinmouth A, Blajchman M A, Kovacs L, Lachance    C, Lee S, Walker C R, Hutton B, Ducharme R, Balchin K, Ramsay T,    Ford J C, Kakadekar A, Ramesh K, Shapiro S. Effect of fresh red    blood cell transfusions on clinical outcomes in premature, very    low-birth-weight infants: the ARIPI randomized trial. JAMA 2012;    308: 1443-1451.-   4. Lacroix J, Hebert P, Fergusson D, Tinmouth A, Blajchman M A,    Callum J, Cook D, Marshall J C, McIntyre L, Turgeon A F. The Age of    Blood Evaluation (ABLE) randomized controlled trial: study design.    Transfus Med Rev 2011; 25: 197-205.-   5. Steiner M E, Assmann S F, Levy J H, Marshall J, Pulkrabek S,    Sloan S R, Triulzi D, Stowell C P. Addressing the question of the    effect of RBC storage on clinical outcomes: the Red Cell Storage    Duration Study (RECESS) (Section 7). Transfus Apher Sci 2010; 43:    107-116.-   6. Hess J R. Red cell changes during storage. Transfus Apher Sci    2010; 43: 51-59.-   7. Hess J R. Red cell storage. J Proteomics 2010; 73: 368-373.-   8. Hod E A, Brittenham G M, Billote G B, Francis R O, Ginzburg Y Z,    Hendrickson J E, Jhang J, Schwartz J, Sharma S, Sheth S, Sireci A N,    Stephens H L, Stotler B A, Wojczyk B S, Zimring J C, Spitalnik S L.    Transfusion of human volunteers with older, stored red blood cells    produces extravascular hemolysis and circulating    non-transferrin-bound iron. Blood 2011; 118: 6675-6682.-   9. Hod E A, Spitalnik S L. Harmful effects of transfusion of older    stored red blood cells: iron and inflammation. Transfusion 2011; 51:    881-885.-   10. Hod E A, Spitalnik S L. Stored red blood cell transfusions:    Iron, inflammation, immunity, and infection. Transfus Clin Biol    2012; 19: 84-89.-   11. Hod E A, Zhang N, Sokol S A, Wojczyk B S, Francis R O, Ansaldi    D, Francis K P, Della-Latta P, Whittier S, Sheth S, Hendrickson J E,    Zimring J C, Brittenham G M, Spitalnik S L. Transfusion of red blood    cells after prolonged storage produces harmful effects that are    mediated by iron and inflammation. Blood 2010; 115: 4284-4292.-   12. Kor D J, Van Buskirk C M, Gajic O. Red blood cell storage    lesion. Bosn J Basic Med Sci 2009; 9 Suppl 1: 21-27.-   13. Silliman C C, Moore E E, Kelher M R, Khan S Y, Gellar L, Elzi    D J. Identification of lipids that accumulate during the routine    storage of prestorage leukoreduced red blood cells and cause acute    lung injury. Transfusion 2011; 51: 2549-2554.-   14. Tissot J D, Rubin O, Canellini G. Analysis and clinical    relevance of microparticles from red blood cells. Curr Opin Hematol    2010; 17: 571-577.-   15. Dumont L J, AuBuchon J P. Evaluation of proposed FDA criteria    for the evaluation of radiolabeled red cell recovery trials.    Transfusion 2008; 48: 1053-1060.-   16. Gilson C R, Kraus T S, Hod E A, Hendrickson J E, Spitalnik S L,    Hillyer C D, Shaz B H, Zimring J C. A novel mouse model of red blood    cell storage and posttransfusion in vivo survival. Transfusion 2009;    49: 1546-1553.-   17. Dominici M, Tadjali M, Kepes S, Allay E R, Boyd K, Ney P A,    Horwitz E, Persons D A. Transgenic mice with pancellular enhanced    green fluorescent protein expression in primitive hematopoietic    cells and all blood cell progeny. Genesis 2005; 42: 17-22.-   18. Klein H G, Anstee D J. Mollisoni's Blood Transfusion in Clinical    Medicine. Malden, Mass: Blackwell Publishing Inc, 2005.-   19. Hoffmann-Fezer G, Maschke H, Zeitler H J, Gais P, Heger W,    Ellwart J, Thierfelder S. Direct in vivo biotinylation of    erythrocytes as an assay for red cell survival studies. Ann Hematol    1991; 63: 214-217.-   20. Soga T, Baran R, Suematsu M, Ueno Y, Ikeda S, Sakurakawa T,    Kakazu Y, Ishikawa T, Robert M, Nishioka T, Tomita M. Differential    metabolomics reveals ophthalmic acid as an oxidative stress    biomarker indicating hepatic glutathione consumption. J Biol Chem    2006; 281: 16768-16776.-   21. Aoki T, Narumiya S. Prostaglandins and chronic inflammation.    Trends Pharmacol Sci 2012; 33: 304-311.-   22. Di Gennaro A, Haeggstrom J Z. The leukotrienes:    immune-modulating lipid mediators of disease. Adv Immunol 2012; 116:    51-92.-   23. Pratico D, Dogne J M. Vascular biology of eicosanoids and    atherogenesis. Expert Rev Cardiovasc Ther 2009; 7: 1079-1089.-   24. Kruckeberg W C, Doorenbos D I, Brown P O. Genetic differences in    hemoglobin influence on erythrocyte oxidative stress hemolysis.    Blood 1987; 70: 909-914.-   25. Dumaswala U J, Wilson M J, Wu Y L, Wykle J, Zhuo L, Douglass L    M, Daleke D L. Glutathione loading prevents free radical injury in    red blood cells after storage. Free Radic Res 2000; 33: 517-529.-   26. Dumaswala U J, Zhuo L, Jacobsen D W, Jain S K, Sukalski K A.    Protein and lipid oxidation of banked human erythrocytes: role of    glutathione. Free Radic Biol Med 1999; 27: 1041-1049.-   27. Dumaswala U J, Zhuo L, Mahajan S, Nair P N, Shertzer H G,    Dibello P, Jacobsen D W. Glutathione protects chemokine-scavenging    and antioxidative defense functions in human RBCs. Am J Physiol Cell    Physiol 2001; 280: C867-C873.-   28. Silliman C C. Lipids: Free Fatty Acids, Eicosanoids, and    Lysophospholipids and the Pro-Inflammatory Effects of Transfusion    ASH Meeting 2012 Scientific Program 2012: SCI-48.-   29. Dern R J, Wiorkowski J J. Studies on the preservation of human    blood. IV. The hereditary component of pre- and poststorage    erythrocyte adenosine triphosphate levels. J Lab Clin Med 1969; 73:    1019-1029.-   30. Mouse Phenome Database [monograph on the internet]. 2012.    Available from: http://phenome.jax.org/

While specific aspects of the invention have been described andillustrated, such aspects should be considered illustrative of theinvention only and not as limiting the invention as construed inaccordance with the accompanying claims.

All publications and patent applications cited in this specification areherein incorporated by reference in their entirety for all purposes asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference for allpurposes.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications can be made thereto without departing from the spiritor scope of the appended claims.

1. A method of determining blood transfusion suitability of a blood sample, the method comprising: measuring levels of markers 9-HODE, 13-HODE, 5-HETE, 12-HETE, and 15-HETE; comparing the levels of the measured markers in the blood sample with levels of corresponding markers present in a control blood sample determined to be suitable for use in a blood transfusion; and using the blood sample in a blood transfusion when an equal or lower level of each measured marker in the blood sample compared to the corresponding marker in the control blood sample is determined; or excluding the blood sample from use in a blood transfusion or discarding the blood sample when a higher level of each measured marker in the blood sample compared to the corresponding marker in the control blood sample is determined.
 2. The method according to claim 1, wherein the method comprises measuring levels of markers 9-HODE, 13-HODE, 5-HETE, 12-HETE, 15-HETE, and arachidonic acid.
 3. The method according to claim 1, wherein the levels of the markers are measured by performing mass spectrometry.
 4. The method according to claim 3, wherein the mass spectrometry is gas-chromatography/mass spectrometry (GC/MS) or liquid chromatography-tandem mass spectrometry (LC/MS/MS).
 5. The method according to claim 1, wherein the levels of the markers are measured by performing an ELISA.
 6. The method according to claim 1, wherein the levels of the markers are measured by performing an enzymatic assay.
 7. The method according to claim 1, wherein the higher level of each marker in the excluded or discarded blood sample is 2-200 fold higher than the corresponding marker in the control blood sample.
 8. The method according to claim 1, wherein comparing the levels of the measured markers further comprises predicting post-transfusion survival of red blood cells of the blood sample based on the compared levels.
 9. The method according to claim 1, wherein 75% or greater of red blood cells in the blood sample used in the blood transfusion are still circulating 24 hours post-transfusion. 